Membrane proteins comprise ~30% of a cell's proteome, and their efficient and accurate localization is essential for the structure and proper functioning of all cells. Compared to the well-studied co-translational protein targeting pathway, post-translational membrane protein targeting poses additional challenges due to the presence of highly hydrophobic transmembrane domains on the substrate protein. Deciphering the molecular strategies to escort such aggregation-prone substrates to the correct target site is a fundamental mechanistic challenge. In the Guided Entry of Tail-anchor (GET) pathway, a complex cascade of protein interactions mediates the post-translational delivery of TA proteins to the endoplasmic reticulum membrane, providing an excellent opportunity to address these questions. Our general goal is to decipher, at the biochemical and biophysical level, the molecular mechanisms underlying the targeting of TA proteins by this novel pathway. Our specific goal is to understand how Get3, the central ATPase in this pathway, uses its ATPase cycle to drive and coordinate the complex cascade of protein interactions during the GET pathway. To this end, we will establish a precise framework for the Get3 ATPase cycle and identify conformational changes that occur during this cycle. We will define when, where and how the upstream and downstream interaction partners of Get3 regulate its ATPase cycle and reciprocally, how this ATPase cycle drives an ordered cascade of interactions of Get3 with its effector proteins. We will develop novel assays to dissect individual steps of the targeting reaction in real time and use this to decipher how highly specific substrate selection is achieved by the pathway. These studies will significantly advance our understanding of the molecular mechanisms that underlie the post- translational targeting of membrane proteins. Further, Get3 represents the first eukaryotic ATPase that belongs to a novel class of 'dimerization-activated' nucleotide hydrolases; studies of this ATPase dimer will be instrumental to test, expand, and generalize the regulatory principles for this growing class of novel cellular regulators.